Mitochondrial Genome Sequences and Structures Aid in the Resolution of Piroplasmida phylogeny

Schreeg, Megan E.; Marr, Henry S.; Tarigo, Jaime L.; Cohn, Leah A.; Bird, David; Scholl, Elizabeth H.; Levy, Michael G.; Wiegmann, Brian M.; Birkenheuer, Adam J.

doi:10.1371/journal.pone.0165702

Cited by 108 publications

(119 citation statements)

References 101 publications

(172 reference statements)

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“…In addition to 18S rRNA, phylogenies based on mitochondrial genes are widely used for the identification of species, including Babesia species (54)(55)(56)(57). Therefore, to further investigate the phylogenetic positions of Babesia sp.…”

Section: Resultsmentioning

confidence: 99%

Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka

et al. 2018

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Bovine babesiosis is a serious threat to the cattle industry. We prepared blood DNA samples from 13 cattle with clinical babesiosis from the Badulla ( = 8), Jaffna ( = 3), and Kilinochchi ( = 2) districts in Sri Lanka. These DNA samples tested positive in PCR assays specific for ( = 9), ( = 9), and ( = 1). Twelve cattle were positive for and/or One cow was negative for the tested species but was positive for on microscopic examination; the phylogenetic positions of 18S rRNA and cytochrome oxidase subunit III gene sequences suggested that the cow was infected with sp. Mymensingh, which was recently reported from a healthy cow in Bangladesh. We then developed a novel sp. Mymensingh-specific PCR assay and obtained positive results for one other sample. Analysis of gene sequences from the cow with positive -specific PCR results demonstrated that the animal was infected not with but with sp. Hue-1, which was recently reported from asymptomatic cattle in Vietnam. The virulence of sp. Hue-1 is unclear, as the cow was coinfected with and However, sp. Mymensingh probably causes severe clinical babesiosis, as it was the sole species detected in a clinical case. The present study revealed the presence of two bovine species not previously reported in Sri Lanka, plus the first case of severe bovine babesiosis caused by a species other than ,, and .

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Section: Resultsmentioning

confidence: 99%

Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka

et al. 2018

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“…Genome-wide phylogenetic analyses indicate that B. microti defines a new clade in the phylum Apicomplexa. Additionally, the difference in both the copy number and organization of multigene families resulted in the phylogeny and life cycle of B. microti being significantly distant from those of other Babesia and Theileria parasites [21,22]. Therefore, the established transfection methods for other apicomplexan parasites may not be used for B. microti.…”

Section: Discussionmentioning

confidence: 99%

Transient Transfection of the Zoonotic Parasite Babesia microti

Liu

Rizk

et al. 2020

Pathogens

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The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5 -actin promoter, program FA100, and 50 µg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.

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“…a a1 Babesia microti (Type-I and Type II orientation) [27] and B. microti -like [28]; b b1 “typical” Piroplasmida [26]…”

Section: Methodsmentioning

confidence: 99%

Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA

et al. 2017

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BackgroundBabesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. Commonly used approaches to diagnose babesiosis include microscopic examination of peripheral blood smears, detection of circulating antibodies and PCR. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene. These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. However, differences in the 18S rRNA gene sequence of distantly related clades can make it difficult to design assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. By targeting Babesia mitochondrial genome (mtDNA), we designed a novel three primer qPCR with greater sensitivity and broader screening capabilities to diagnose and differentiate Babesia spp.MethodsUsing 13 Babesia mtDNA sequences, a region spanning two large subunit rRNA gene fragments (lsu5-lsu4) was aligned to design three primers for use in a qPCR assay (LSU qPCR) capable of amplifying a wide range of Babesia spp. Plasmid clones were generated and used as standards to determine efficiency, linear dynamic range and analytical sensitivity. Animals naturally infected with vector-borne pathogens were tested retrospectively and prospectively to determine relative clinical sensitivity and specificity by comparing the LSU qPCR to an established 18S rDNA qPCR.ResultsThe LSU qPCR efficiencies ranged between 92 and 100% with the limit of detection at five copies/reaction. The assay did not amplify mammalian host or other vector-borne pathogen gDNA except Cytauxzoon felis (a feline protozoal pathogen). The LSU qPCR assay amplified 12 different Babesia. sp. and C. felis from 31/31 (100%) archived samples, whereas the 18S qPCR amplified only 26/31 (83.9%). By prospective analysis, 19/394 diagnostic accessions (4.8%) were LSU qPCR positive, compared to 11/394 (2.8%) 18S rDNA qPCR positive.ConclusionsWe have developed a more sensitive qPCR assay with a more expansive range of Babesia spp. detection by targeting a highly conserved region of mtDNA, when compared to an established 18S qPCR.

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Mitochondrial Genome Sequences and Structures Aid in the Resolution of Piroplasmida phylogeny

Cited by 108 publications

References 101 publications

Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka

Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka

Transient Transfection of the Zoonotic Parasite Babesia microti

Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA

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